Redox homeostasis and inflammation in fibroblasts of patients with Friedreich Ataxia: a possible cross talk

Abstract

Redox homeostasis is impaired in Friedreich's Ataxia (FRDA), a neurodegenerative disease caused by the decreased expression of the mitochondrial protein frataxin. Nrf2, the master regulator of tissue redox balance, is defective in the disease, driving cells to ferroptosis. Neuro-inflammation is recently emerging as an additional pathological mechanism in FRDA and has to be understood in order to go deeper into the pathogenesis of the disease. As a functional cross talk between Nrf2 and NF-kB pathways has been previously reported, we wonder if inflammation may be activated in FRDA as a consequence of Nrf2 deficiency. Thus, we analyzed the expression of proteins involved in the antioxidant and inflammatory responses in fibroblasts of patients with FRDA. We found a significant activation of the TLR4/NF-kB/IL-1β axis in patients, associated to a consistent increase of the redox enzymes thioredoxin 1 (TRX1) and glutaredoxin 1 (GLRX1), which are essential to activate NF-kB under oxidative stress conditions. Furthermore, we investigated the role of 4-HNE, a by-product of lipid peroxidation, as a potential mediator between ferroptosis and inflammation in FRDA.

Keywords: Freidreich Ataxia; GLRX1; IL-1β; NF-kB; Nrf2; TLR4; Trx1.

Figure 1

Densitometry of NF-kB p65 (A) and IL-1β (B) protein levels in fibroblasts of n.3 patients with FRDA vs. n.3 control cells (CTRLs), as determined by Western blot analysis. (C) Representative Western blot images of NF-kB p65, IL-1β and GAPDH. (D) IL-1β content as measured by ELISA in FRDA cells. Values are expressed as mean ± SEM. Statistical significance was determined by Student’s t test and defined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 respect to controls.

Figure 2

mRNA (A,B) and protein (C,D) levels of TRX1 and GLRX1 in n.3 FRDA fibroblasts and n.3 control subjects, as determined by qRT-PCR and Western blot analysis, respectively. (E) Representative Western blot of TRX1, GLRX1, and GAPDH (as loading control). Data are expressed as mean ± SEM. Statistical significance was determined by Student’s t test and defined as *p < 0.05, **p < 0.01, ***p < 0.001 respect to controls.

Figure 3

qRT-PCR analysis of toll-like receptor 4 (TLR4) expression in n. 3 FRDA fibroblasts and n. 3 controls (CTRLs). Values are expressed as mean ± SEM. Statistical significance was determined by Student’s t test and defined as ****p < 0.0001 respect to controls.

Figure 4

(A) TLR4 and NF-kB p65 mRNA expression upon RSL-3 treatment, as determined by qRT-PCR. (B) Western blot analysis of NF-kB p65 and IL-1β protein amounts, along with representative Western blot images of the respective proteins. Values are expressed as mean ± SEM. Statistical analyses were performed by ANOVA and significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 respect to controls.

Figure 5

mRNA (A) and protein levels (B) of TRX1 and GLRX1 in control fibroblasts after RSL-3 treatment. A representative Western blot of TRX1 and GLRX1 is also reported. Values are expressed as mean ± SEM. Statistical analyses were performed by ANOVA and significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 respect to controls.

Figure 6

4-HNE levels, as measured by ELISA, in n.3 control fibroblasts incubated for 3 h and 5 h with 250 nM RSL3 (A) and in FRDA fibroblasts from n.3 patients (B). Cells were used at similar passage numbers (9–11) and the assays performed in triplicates. Values are expressed as mean ± SEM. Statistical significance was determined by Student’s t test and defined as *p < 0.05 and **p < 0.01.